Codelivery of BCL2 and MCL1 Inhibitors Enabled by Phenylboronic Acid‐Functionalized Polypeptide Nanovehicles for Synergetic and Potent Therapy of Acute Myeloid Leukemia

Abstract Acute myeloid leukemia (AML) is the most refractory hematologic malignancy characterized by acute onset, rapid progression, and high recurrence rate. Here, codelivery of BCL2 (ABT199) and MCL1 (TW37) inhibitors using phenylboronic acid‐functionalized polypeptide nanovehicles to achieve synergetic and potent treatment of AML is adopted. Leveraging the dynamic boronic ester bonds, B—N coordination, and π–π stacking, the nanovehicles reveal remarkably efficient and robust drug coencapsulation. ABT199 can induce a series of pro‐apoptotic reactions by promoting the dissociation of the pro‐apoptotic protein Bim from BCL2, while the released Bim is often captured by MCL1 protein overexpressed in AML. TW37 has a strong inhibitory ability to MCL1, thereby can restrain the depletion of Bim protein. Dual inhibitor‐loaded nanoparticles (NPAT) reveal excellent stability, acid/enzyme/H2O2‐triggered drug release, and significant cytotoxicity toward MOLM‐13‐Luc and MV‐411 AML cells with low half maximal inhibitory concentrations of 1.15 and 7.45 ng mL−1, respectively. In mice bearing MOLM‐13‐Luc or MV‐411 AML cancer, NPAT reveal significant inhibition of tumor cell infiltration in bone marrow and main organs, potent suppression of tumor growth, and remarkably elevated mouse survival. With facile construction, varying drug combination, superior safety, synergetic efficacy, the phenylboronic acid‐functionalized smart nanodrugs hold remarkable potential for AML treatment.

S3 C) following a guard column and a differential refractive-index detector. The measurements were performed using DMF (containing 10 mM LiBr) as the eluent at a flow rate of 1.0 mL/min at 30 o C and a series of narrow polystyrene standards for the calibration of the columns. The sizes of nanoparticles were determined using dynamic light scattering (DLS). Measurements were carried out at 25 o C by Zetasizer Nano-ZS from Malvern Instruments equipped with 633 nm He-Ne laser using backscattering detection. The zeta potential of nanoparticles was determined with Zetasizer Nano-ZS from Malvern Instruments equipped with a standard capillary electrophoresis cell. The drug concentrations of TW37 and ABT199 were detected by high performance liquid chromatograph (HPLC). The column temperature was set at 30 ºC, the flow rate was 1.0 mL/min, the injection volume was 10 μL, the UV detection wavelength was 300 nm, and the mobile phase was acetonitrile/ water (80/20, v/v).

Alizarin Red S Competitive Binding Experiment
In order to verify that the boron ester bonds formed between PEG-b-P(BPA-co-Tyr) and TW37, alizarin red S (ARS) competitive binding experiment was performed. ARS with no fluorescence can combine with boronic acid moieties to form ARS complexes that often exhibit strong fluorescence. The fluorescence intensity of formed ARS complexes would decrease S4 through competitive binding with substances containing cis-1,2-or 1,3-diols. Briefly, a PEGb-P(BPA-co-Tyr) solution (95.2 μg/mL, 0.5 mL) was thoroughly mixed with ARS solution (68.4 μg/mL, 0.5 mL) for 3 min. Then, predetermined amount (0, 1.44 μL, 2.87 μL, 4.31 μL, 5.74 μL, 14.4 μL, 28.7 μL) of TW37 (20 mg/mL) dissolved in DMSO was added to the mixed solution. After stirring with a shaker (37 o C, 100 rpm) for 10 min, the fluorescence intensity at 520-720 nm was detected using a fluorophotometer with an excitation wavelength at 470 nm.

In Vitro Triggered Drug Release
In vitro drug release was carried out in four different media (25 mL): (i) PBS (150 mM, pH 7.4), (ii) PBS (150 mM, pH 5.5), (iii) PBS (150 mM, pH 7.4) containing proteinase K (PK, 12 U/mL), and (iv) PBS (150 mM, pH 7.4) containing 100 μM H2O2. NPAT (0.5 mL, 0.5 mg/mL) added in a dialysis tube (MWCO 30 kDa) was immersed in different release media under shaking at 37 °C. At predetermined time points, 5.0 mL of release medium was withdrawn and refilled with fresh medium. The amount of TW37 and ABT199 in release medium was quantified using high performance liquid chromatography (HPLC). The cumulative drug release was calculated according to the following formula: Er: cumulative release of TW37 or ABT199 (%); Ve: displacement volume of release medium (5.0 mL); V0: total volume of release medium (25 mL); Ci: release medium at the i-th sampling concentrations of TW37 or ABT199 (μg/mL); mdrug: total amount of TW37 or ABT199 in NPAT used for release (μg); n: number of replacement medium.

Mitochondrial Membrane Permeability
To analyze the mitochondrial membrane permeability of MOLM-13-Luc and MV-411 cells, the cells in 6-well plate (5 × 10 5 cells/well) were incubated with NPT, NPA or NPAT for 48 h. The drug concentration was 10 ng/mL for MOLM-13-Luc cells and 60 ng/mL for MV-S5 min according to the user's manual of mitochondrial membrane potential assay kit. Then, the cells were subjected to flow cytometry analysis, and the ratios of the intact mitochondria to damaged mitochondria as compared to that of PBS group were calculated.

Immunohistochemical Analysis
The tissue was formalin-fixed, paraffin-embedded, sectioned (5 μm thickness), and mounted onto microscope slides. After deparaffinizing and rehydrating, the tissue sections were sequentially incubated with primary antibody against human CD45 (Abcam, UK) and biotinylated secondary antibody (ZSGB-Bio, China). Tyramide signal amplification (TSA) technology was used for fluorescence color rendering. Images were taken using a fluorescence microscope system under the same magnification.